U.S. patent application number 17/046671 was filed with the patent office on 2021-02-04 for bispecific antibody with prolonged half-life and enhanced anti-tumor effect.
The applicant listed for this patent is SOUND BIOPHARMACEUTICALS CO. LTD.. Invention is credited to Fanxin MA.
Application Number | 20210032362 17/046671 |
Document ID | / |
Family ID | 1000005193555 |
Filed Date | 2021-02-04 |
![](/patent/app/20210032362/US20210032362A1-20210204-D00001.png)
![](/patent/app/20210032362/US20210032362A1-20210204-D00002.png)
![](/patent/app/20210032362/US20210032362A1-20210204-D00003.png)
![](/patent/app/20210032362/US20210032362A1-20210204-D00004.png)
![](/patent/app/20210032362/US20210032362A1-20210204-D00005.png)
![](/patent/app/20210032362/US20210032362A1-20210204-D00006.png)
![](/patent/app/20210032362/US20210032362A1-20210204-D00007.png)
United States Patent
Application |
20210032362 |
Kind Code |
A1 |
MA; Fanxin |
February 4, 2021 |
BISPECIFIC ANTIBODY WITH PROLONGED HALF-LIFE AND ENHANCED
ANTI-TUMOR EFFECT
Abstract
Disclosed is a PEGylated bispecific antibody, comprising (a) an
antigen-binding fragment (Fab), which has a light chain variable
region (VL) and a light chain constant region (CL), a heavy chain
Variable region (VH) and a part of the heavy chain constant region
(CH1); (b) a single domain antigen binding fragment (VHH) fused to
the C-terminus of the part of the heavy chain constant region (CH1)
of the antigen binding fragment (Fab), and (c) Polyethylene glycol
(PEG) fused to the C-terminus of the light chain constant region
(CL) of the antigen-binding fragment (Fab). The PEGylated S-Fab
(PEG-S-Fab) retained the binding specificity to both tumor cells
and T cells. PEG-S-Fab enhanced the plasma stability and had a
12-fold increased half-life of S-Fab. PEG-S-Fab also had more
potent tumor inhibiting efficacy in xenograft mouse model. Those
data suggest that PEGylation can be an effective approach to
enhance anti-tumor properties of bispecific antibodies.
Inventors: |
MA; Fanxin; (Chengdu,
CN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
SOUND BIOPHARMACEUTICALS CO. LTD. |
Chengdu |
|
CN |
|
|
Family ID: |
1000005193555 |
Appl. No.: |
17/046671 |
Filed: |
April 3, 2019 |
PCT Filed: |
April 3, 2019 |
PCT NO: |
PCT/CN2019/081164 |
371 Date: |
October 9, 2020 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 2317/569 20130101;
C07K 2317/55 20130101; C07K 2317/515 20130101; C07K 2317/73
20130101; C07K 2317/51 20130101; C07K 16/2809 20130101; A61K
2039/505 20130101; C07K 2317/31 20130101; A61P 35/00 20180101; C07K
2317/94 20130101; C07K 16/3007 20130101 |
International
Class: |
C07K 16/30 20060101
C07K016/30; C07K 16/28 20060101 C07K016/28; A61P 35/00 20060101
A61P035/00 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 9, 2018 |
CN |
201810310970.X |
Claims
1. A PEGylated bispecific antibody, comprising (a) an
antigen-binding fragment (Fab) having a light chain variable region
(VL) and a light chain constant region (CL), a heavy chain variable
region (VH) and a part of a heavy chain constant region (CH1); (b)
a single domain antigen-binding fragment (VHH) fused to a
C-terminus of the part of the heavy chain constant region (CH1) of
the antigen binding fragment (Fab); and (c) polyethylene glycol
(PEG) fused to a C-terminus of the light chain constant region (CL)
of the antigen-binding fragment (Fab).
2. The PEGylated bispecific antibody of claim 1, wherein the
polyethylene glycol is connected to the C-terminus of the light
chain constant region (CL) through at least one cysteine residue at
the C-terminus of the light chain constant region (CL).
3. The PEGylated bispecific antibody of claim 2, wherein when the
polyethylene glycol is connected to the C-terminus of the light
chain constant region (CL) through two cysteine residues at the
C-terminus of the light chain constant region (CL), a linker
sequence is spaced between the cystine residues.
4. The PEGylated bispecific antibody of claim 1, wherein the
molecular weight of the polyethylene glycol is 10,000 to
30,000.
5. The PEGylated bispecific antibody of claim 4, wherein the
molecular weight of the polyethylene glycol is 20,000.
6. The PEGylated bispecific antibody of claim 1, wherein the
antigen-binding fragment is specific for tumor antigens, and the
single domain antigen-binding fragment is specific for immune
cells.
7. The PEGylated bispecific antibody of claim 6, wherein the tumor
antigen is selected from a group consisting of CEA, EGFR, Her2,
EpCAM, CD20, CD30, CD33, CD47, CD52, CD133, CEA, gpA33, Mucin,
TAG-72, CIX, PSMA, folate binding protein, GD2, GD3, GM2, VEGF,
VEGFR, integrin, .alpha.V.beta.3, .alpha.5.beta.1, ERBB2, ERBB3,
MET, IGF1R, EPHA3, TRAILR1 TRAILR2, RANKL, FAP and Tenascin.
8. The PEGylated bispecific antibody of claim 7, wherein the tumor
antigen is CEA or Her2.
9. The PEGylated bispecific antibody of claim 6, wherein the single
domain antigen-binding fragment is specific for mammalian T cells
or mammalian NK cells.
10. The PEGylated bispecific antibody of claim 6, wherein the
single domain antigen binding fragment has specificity for any
antigen selected from the group consisting of CD3, CD16, CD19, CD28
and CD64.
11. The PEGylated bispecific antibody of claim 10, wherein the
antigen is CD16 or CD3.
12. An engineered bispecific antibody comprising (a) an
antigen-binding fragment (Fab) having a light chain variable region
(VL) and a light chain constant region (CL), a heavy chain variable
region (VH) and a part of a heavy chain constant region (CH1); and
(b) a single domain antigen binding fragment (VHH) fused to the
C-terminus of the part of the heavy chain constant region (CH1) of
the antigen binding fragment (Fab); wherein the C-terminus of the
light chain constant region (CL) of the antigen-binding fragment
(Fab) is engineered to have one or two cysteine residues.
13. The engineered bispecific antibody of claim 12, wherein when
there are two cysteine residues, there is a linker sequence between
the two cysteine residues.
14. (canceled)
15. A pharmaceutical composition comprising the bispecific antibody
of claim 1 and a pharmaceutically acceptable carrier.
Description
TECHNICAL FIELD
[0001] The present invention relates to bispecific antibodies, and
in particular to PEGylated bispecific antibodies with prolonged
half-life and enhanced anti-tumor effect.
BACKGROUND
[0002] Bispecific antibody has been developed as a powerful
approach in cancer immunotherapy by engaging immune cells directly
to the cancer cells. Bispecific antibodies have two different
antigen binding sites with one recognizing the tumor cells and the
other recognizing the immune cells, usually T cells or natural
killer (NK) cells. Various bispecific antibody formats have been
studied, including IgG based full-length formats (such as the
complete bispecific antibody), single chain-based formats
(including tandem single-chain variable fragments (ScFv)) and
bispecific T-cell engager (BiTE).
[0003] In order to enhance the tumor tissue penetration of
full-size IgG antibodies, and improve the stability of ScFv and
BiTE, the bispecific single domain antibody-linked Fab (S-Fab)
format was developed, which can bind to diverse epitopes and use
prokaryotic expression systems. Derived from the natural camel
heavy-chain only antibodies, single domain antibodies lack the
first constant (CH1) domain and light chain, and are consequently
referred to as nanobodies or VHHs. Nanobodies are small-sized and
generally more stable than conventional scFv or BiTE, making them
good specialty for constructing bispecific antibodies. In a
previous study, a bispecfic S-Fab antibody was constructed by
linking the single-domain nanobody anti-CEA to an CD3-Fab. The
S-Fab exhibited excellent tumor cell killing activities in vitro.
(L. Li, P. He, C. Zhou, L. Jing, B. Dong, S. Chen, N. Zhang, Y.
Liu, J. Miao, Z. Wang, Q. Li, A novel bispecific antibody, S-Fab,
induces potent cancer cell killing, Journal of immunotherapy, 38
(2015) 350-356).
[0004] However, Fab fragments (including S-Fab) still have short
plasma half-lives as a result of rapid degradation in vivo, due to
the lack of the constant regions (Fc), which makes them less ideal
for long-term clinical use.
SUMMARY
[0005] In one aspect, provided is a PEGylated bispecific antibody
(also called PEG-S-Fab herein), comprising (a) an antigen-binding
fragment (Fab) having a light chain variable region (VL) and a
light chain constant region (CL), as well as a heavy chain variable
region (VH) and a part of a heavy chain constant region (CH1); (b)
a single domain antigen-binding fragment (VHH) fused to the
C-terminus of the part of the heavy chain constant region (CH1) of
the antigen binding fragment (Fab); and (c) polyethylene glycol
(PEG) fused to the C-terminus of the light chain constant region
(CL) of the antigen-binding fragment (Fab). The molecular weight of
PEG suitable for use in the present disclosure can be determined
according to the routine test in the field. In some embodiments,
the molecular weight of the polyethylene glycol is 10,000 to
30,000. In another embodiments, the molecular weight of the
polyethylene glycol is 20,000.
[0006] The present invention also provides a pharmaceutical
composition comprising the PEGylated bispecific antibody, and a
pharmaceutically acceptable carrier or excipient. Correspondingly,
the present invention provides a host cell comprising one or more
polynucleotides encoding the bispecific antibody.
[0007] In another aspect, provided is an engineered bispecific
antibody comprising (a) an antigen-binding fragment (Fab) having a
light chain variable region (VL) and a light chain constant region
(CL), a heavy chain variable region (VH) and a part of a heavy
chain constant region (CH1); and (b) a single domain antigen
binding fragment (VHH) fused to the C-terminus of the part of the
heavy chain constant region (CH1) of the antigen binding fragment
(Fab); wherein the C-terminus of the light chain constant region
(CL) of the antigen-binding fragment (Fab) is engineered to have at
least one cysteine residue. Correspondingly, the present invention
provides a use of the engineered bispecific antibody in preparing
the PEGylated bispecific antibody of the present invention.
[0008] Another aspect of the present invention provides a method of
treating cancer, comprising contacting the PEGylated bispecific
antibody of the present invention with cancer cells. Accordingly,
the present invention provides a method of treating cancer in a
subject, which comprises administering to the subject a
therapeutically effective amount of the PEGylated bispecific
antibody of the present invention.
[0009] Another aspect of the present invention provides a host cell
comprising one or more polynucleotides encoding the bispecific
antibody as described above. In some embodiments, the host cell is
E. coli. The manipulation of polynucleotides involves knowledge and
experimental operations in the fields of molecular biology, genetic
engineering, protein engineering, etc., which are well known to
those skilled in the art.
[0010] The inventors explored a thiol site-specific PEGylation to
improve the half-life (a2) of S-Fab bispecific antibody. In this
study, a functionalized 20 kDa linear methoxy PEG maleimide
(MAL-PEG-OMe) was used to conjugate S-Fab. The PEGylated S-Fab
(PEG-S-Fab) retained the binding specificity to both tumor cells
and T cells. PEG-S-Fab enhanced the plasma stability and had a
12-fold increased half-life of S-Fab. PEG-S-Fab also had more
potent tumor inhibiting efficacy in xenograft mouse model. Those
data suggest that PEGylation can be an effective approach to
enhance anti-tumor properties of bispecific antibodies.
BRIEF DESCRIPTION OF DRAWINGS
[0011] FIG. 1 Expression and purification of S-Fab from E. coli. A,
The bacterial S-Fab expression constructs contain a pelB signal
sequence, an anti-CD3 (human UTCH1 clone) VH (or VL) and CH (CL),
and an anti-CEA-VHH. To facilitate antibody detection and
purification, a flag-tag and a his6-tag were added to the
C-terminal end of heavy and light chain, respectively; B, Schematic
presentation of S-Fab after co-expression; C, Coomassie
blue-stained SDS-PAGE of the purified S-Fab after the two-step
purification; D, Gel filtration analysis showed that the molecular
weight of S-Fab was approximately 130 kD.
[0012] FIG. 2 PEGylation of bispecific S-Fab using 20 kDa linear
methoxy PEG maleimide (MAL-PEG-OMe). S-Fab reacted with the
functionalized PEG at a series of ratios (0:1, 10:1, 20:1, 40:1 and
60:1) mol equiv of PEG:S-Fab and shaking at 22.degree. C. for 2
hrs, the samples were then examined with SDS-PAGE (5 ul/sample). A.
Coomassie blue staining for the S-Fab with different molar ratio of
PEG:S-Fab in PEGylation process. B. Barium iodide complex dye
staining for the S-Fab with different molar ratios of PEG:S-Fab. C.
Western blot using anti-flag antibody to detect the VH--CH-CEA
chain. D. Western blot using anti-his6 antibody to detect the VL-CL
chain. Note: 0, 10, 20, 40 and 60 represent as 0:1, 10:1, 20:1,
40:1 and 60:1 for the molar ratio of PEG:Fab.
[0013] FIG. 3 Purification of PEG-S-Fab. The gel filtration was
used to fraction the PEGylation mixture (Fractions 1, 2, 3). A.
Chromatogram of PEG-S-Fab. B. Coomassie blue staining for the
fractions of PEG-S-Fab purification. C. Barium iodide complex
staining for the fractions of PEG-S-Fab purification.
[0014] FIG. 4 PEG-S-Fab can bind CEA on tumor cells and CD3.sup.+
on T cells. Flow cytometry analysis with PEG-S-Fab and S-Fab was
performed on CEA-positive LS174T cells (A), CEA-negative SKOV3
cells (B), and CD3.sup.+ T cells (C). Positive control anti-CD3
antibody OKT3 was used for T cell flow cytometry. Confocal
microcopy of immunofluorescence staining of S-Fab (D) and PEG-S-Fab
(E) in LS174T cells (upper panel) and SKOV3 cells (lower panel),
respectively. The bar scale represents 30 .mu.m.
[0015] FIG. 5 PEG-S-Fab has potent specific cytotoxicity against
tumor cells. A. The cytotoxic assay using CEA-positive LS174T
cells. B. The cytotoxic assay using CEA-negative SKOV3 cells. The
cytotoxic assays were performed as described in section 2.8.
Different concentrations of S-Fab or PEG-S-Fab were incubated with
tumor cells and effector T cells (E/T=10). All data are showed as
the mean of triplicates, with error bars representing the SD.
[0016] FIG. 6 Pharmacokinetic and stability analysis of serum
concentrations of PEG-S-Fab and S-Fab. A. Standard curve for the
quantified ELISA. B. The serum protein concentration-time curve
after intravenous administration. (Note: Each data point was
expressed as the mean.+-.SEM). C-D. Western blotting to detect the
resulting S-Fab (C) and PEG-S-Fab (D) using anti-flag antibody.
E-F. Western blotting to detect the resulting S-Fab (C) and
PEG-S-Fab (D) using anti-his antibody. (Note: M, P--S--Fab and P
represent as marker, PEG-S-Fab and plasma only, respectively).
[0017] FIG. 7 PEGylation of S-Fab induces more potent in vivo
anti-tumor activity. B-NDG mice (n=6 per group) were subcutaneously
engrafted with LS174T cells and human PBMCs. The mice were then
treated with PBS alone, 0.3 nmol of S-Fab or S-Fab-PEG20K daily
over six days. The data represent the average tumor volume of six
mice. The error bars represent the SEM. Data were analyzed with
two-way ANOVA analysis using GraphPad Prism 5 software, and ***
represents as p<0.01 when compared the vehicle with S-Fab or
PEG-S-Fab groups.
DETAILED DESCRIPTION OF THE INVENTION
Definition
[0018] It should be noted that the term "a" or "an" entity refers
to one or more of that entity, for example, "a bispecific antibody"
is understood to represent one or more bispecific antibodies.
Likewise, the terms "a", "one or more" and "at least one" are used
interchangeably herein.
[0019] The term "antibody" or "antigen-binding polypeptide" as used
herein refers to a polypeptide or polypeptide complex that
specifically recognizes and binds to one or more antigens.
Antibodies are complete antibodies or any antigen-binding fragments
or single chains thereof. Therefore, the term "antibody" includes
any protein or peptide that contains at least a part of an
immunoglobulin molecule that has the biological activity of binding
to an antigen. Such examples include, but are not limited to, the
complementarity determining region (CDR) of the heavy/light chain
or its ligand binding portion, the variable region of the heavy or
light chain, the constant region of the heavy or light chain, and
the framework (FR) region or any part thereof, or at least part of
a binding protein. The term antibody also encompasses polypeptides
or polypeptide complexes that have antigen-binding ability once
activated. In some examples, for example, certain immunoglobulin
molecules derived from or based on the engineering of camelid
immunoglobulins. The whole immunoglobulin molecule may only consist
of heavy chains without light chains, see for example
Hamers-Casterman et al., Nature 363:446-448 (1993).
[0020] The term "specific binding" or "specific to" generally means
that the antibody binds to the epitope through its antigen binding
domain, and the binding requires a certain complementarity between
the antigen binding domain and the epitope. According to this
definition, when an antibody can bind to a specific epitope more
rapidly via its antigen binding domain than to a random unrelated
epitope, the antibody is said to "specifically bind" to that
epitope. The term "specificity" is used to quantify the relative
affinity of a specific antibody to a specific epitope. For example,
for a given epitope, antibody "A" is considered to have a higher
specificity than antibody "B", or antibody "A" binds to epitope "C"
more specifically than it binds to related epitope "D".
[0021] The term "treatment" as used herein refers to a therapeutic
or preventive approach in which a subject is prevented from or
slowed down (alleviated) an undesirable pathological change or
disorder, such as the progression of cancer. Beneficial or desired
clinical results include, but are not limited to, alleviation of
symptoms, reduction of disease severity, stabilization of the
disease state (that is, no deterioration), delay or slowing of
disease progression, improvement or alleviation of disease state,
and alleviation (either locally or systematically), detectable or
undetectable. The term "treatment" also refers to prolonged
survival compared to expected survival if not receiving the
treatment. Subject in need of treatment include those who already
have a disease or condition, as well as those who tend to have the
disease or condition or those who need to prevent the disease or
condition.
[0022] "Subject" or "individual" or "animal" or "patient" or
"mammal" refers to any subject for which diagnosis, prognosis, or
treatment is desired, especially a mammalian subject. Mammal
subjects include humans, domestic animals, farm animals, zoo
animals, sports animals or pets, such as dogs, cats, guinea pigs,
rabbits, rats, mice, horses, bovines, cows, and the like.
[0023] The term "patient in need of treatment" or "subject in need
of treatment" includes subjects who can benefit from the
administration of the antibody or composition of the present
invention, such as mammalian subjects, for purposes of such as
detection, diagnostic procedures and/or treatment.
[0024] As confirmed in the examples, an exemplary bispecific
antibody is an antibody that targets two different antigens, one of
which is present on tumor cells or microorganisms, and the other is
on immune cells. When administered to an individual, the bispecific
antibody specifically binds to tumor cells or microorganisms, and
at the same time specifically binds to immune cells (such as
cytotoxic cells). This dual binding can cause the bound tumor or
microorganism to be killed by the host's immune system.
[0025] A "single domain antigen-binding fragment" or "single domain
antibody fragment" or "VHH" is an antigen-binding fragment capable
of binding to an antigen without being equipped with a light chain.
VHH was originally isolated from a single domain antibody (sdAb) as
a single antigen-binding fragment. The first known single domain
antibody was isolated from camel (Hamers-Casterman et al., Nature
363:446-8 (1993)) and later from cartilaginous fish. Camels produce
functional antibodies without light chains, and their single
N-terminal domain (VHH) binds to antigen without domain pairing
(see Harmsen and Haard, App Microbiol Biotechnol, 77:13-22 (2007)
for review). Single domain antibodies do not include the CH1 domain
which, in conventional antibodies, interacts with the light
chain.
[0026] VHH contains four framework regions (FR1-FR4) constituting
the core structure of an immunoglobulin domain and three
complementarity determining regions (CDR1-CDR3) involved in antigen
binding. Compared with the human VH domain, the VHH framework
region shows high sequence homology (>80%) with the human VH
domain. See Harmsen and Haard, 2007, which further describes: "the
most characteristic feature of VHH lies in the amino acid
substitutions at the four FR2 positions (positions 37, 44, 45 and
47; Kabat numbering), which are conserved and involved in
hydrophobic interactions with the VL domain in conventional VH
structures". VHH usually has different amino acids at these and
other positions that are highly conserved in conventional VH (such
as LeulISer, Val37Phe or Tyr, Gly44Glu, Leu45Arg or Cys,
Trp47Gly).
[0027] Harmsen and Haard, 2007 also described: VHH CDR has some
known characteristic features. For example, the N-terminal part of
CDR1 is more variable than conventional antibodies. Moreover,
certain VHHs have a next ended CDR3 which is usually stabilized by
forming additional disulfide bonds with the cysteine in CDR1 or
FR2, resulting in the CDR3 loop folding over the entire interface
of the previous VL. The specific subfamily of llama VHH (VHH3) also
contains an extended CDR3 which is stabilized by forming an
additional disulfide bond width cysteine at position 50 of FR2.
[0028] Many single domain antibodies (sdAbs) are known in the art
and can be easily prepared from animals such as camels. Based on
these sdAbs, their VHH can be easily identified and prepared. Table
1 lists many non-limiting examples of VHH and sdAb. Therefore, in
some embodiments, the present invention provides a polypeptide
comprising each such disclosed sequence or its equivalent and a
polynucleotide encoding each polypeptide.
TABLE-US-00001 TABLE 1 Exemplary single domain antigen binding
fragments (VHH) and single domain antibodies (sdAb) 1. anti-CEA VHH
(SEQ ID NO: 1) EVQLVESGGG FVQAGESLTL SCTSSTLTFT PYRMAWYRQA
PGKQRDLVAD ISSGDGRTTN YADFAKGRFT ISRDNIKNTV FLRMTNLKPE DTAVYYCNTF
VSFVGIARSW GQGTQVTVSS 2. anti-CD16 VHH (SEQ. ID NO: 2) EVQLVESGGG
LVQPGGSLRL SCSFPGSIFS LTMGWYRQAP GKERELVTSA TEGGDTNYAD FVKGRFTISR
DNARSIIYLQ MNSLKPEDTA VYYCYARTRN WG 3. anti-CD16 VHH (SEQ ID NO: 3)
EVQLVESGGE LVQAGGSLRL SCAASGLTFS SYNMGWFRRA PGKEREFVAS ITWSGRDTFY
ADSVKGRFTI SRDNAKNTVY LQMSSLKPED TAVYYCAANP WP 4. anti-CD16 VHH
(SEQ ID NO: 4) EVQLVESGGG LVQEGESLTL SCVVAGSIFS FAMSWYRQAP
GKERELVARI GSDDRVTYAD SVKGRFTISR DNIKRTAGLQ MNSLKPEDTA VYYCNAQTDL
RD 5. anti-CD16 VHH (SEQ ID NO: 5) EVQLVESGGG LVQPGGSLTL SCVAAGSIFT
FAMSWYRQAP RKERELVARI GTDDETMYKD SVKGRFTISR DNVKRTAGLQ MNNLKPEDTA
VYYCNARTDY RD 6. anti-Her2 sdAb (SEQ ID NO: 6) QVQLVQSGGG
LVQAGGSLRL SCAASGRTFS SYAMAWFRQA PGKEREFVAA ISWSGANIYV ADSVKGRFTI
SRDNAKDTVY LQMNSLKPED TAVYYCAVKL GFAPVEERQY DYWGQGTQVT VSS 7.
anti-EGFR1 VHH (SEQ ID NO: 7) MAEVQQASGG GLVQAGGSLR LSCAASGRTE
TTSAIAWFRQ APGKEREFVA QISASGLGIN YSGTVKGRFT ISRDADKTTV YLQMNSLTPE
DTAVYYCAAG FHYIAAIRRT TDFHFWGPGT LVTVSSGR 8. anti-F4 + ETEC
bacteria VHH (SEQ ID NO: 8) QVQLQESGGG LVQAGGSLRL SCEASGNVDR
IDAMGWERQA PGKQREFVGY ISEGGILNYG DEVKGRETIS RDNAKNTVYL QMSNLKSEDT
GVYFCAASHW GTLLIKGTEH WGKGTQVTVS S 9. anti-PS2-8 VHH (SEQ ID NO: 9)
EVQLVESGGG LVQAGGSLRL SCAASGRSFS RDAMGWFRQA PGKERDVVAA INLNGGRTYS
ADSVKGPFTI SRDNDKNTVY LQMSNLKPED TAVYYCAARE GDVGLVSYKR SSNYPYWGQG
TQVTVSS 10. anti-Huntavirus VHH (SEQ ID NO: 10) MAEVQLQASG
GGLVQAGGSL RLSCAASGRT SSMYSMVWFR QAPGKEREFV AGIIWTSSLT YYADSLKGRF
TISRDNAKNT VYLQMNSLKP EDTAIYYCAA DTKTGGGGYE YWGQVTVTVS S 11.
anti-Huntavirus VHH (SEQ ID NO: 11) MAEVQLQASG GGLVQPGGSL
RLSCAASGSI FSSDVMGWFR QAPGKERELV AFITDDGGTN YADSVKGRFT ISRDNAENTV
SLQMNSLKPE DTAVYYCNAR YYSGGYRNYW GQVTVTVSS 12. anti-CD16 VHH (SEQ
ID NO: 12) EVQLVESGGG LVQPGGSLRL SCSFPGSIFS LTMGWYRQAP GKERELVTSA
TPGGDTNYAD FVKGRETISR DNARSIIYLQ MNSLKPEDTA VYYCYARTPN
WGTVWGQGTQVTVSS 13. anti-CD3 VHH (SEQ ID NO: 13) GVQLQESGGG
LVQAGGSLRL SCAASGRTFS NYHMGWFRQA PGKERELVAA ISGSGGSTYY TDSVFGRFTI
SPNNAKNTMS LQMSNLKPED TGVYYCTTPT EKGSSIDYWG QGTQVTVSSG
RYPYDVPDY
[0029] In some embodiments, the Fab or VHH (or VHH') fragment of
the bispecific antibody has immunologic specificity to tumor
antigens.
[0030] "Tumor antigen" is anantigenic substance produced in tumor
cells, that is, it causes an immune response from the host. Tumor
antigens can be used to identify, tumor cells and are potential
candidates for cancer treatment. The normal protein in the body is
not antigenic. However, certain proteins are produced or
overexpressed during the tumorigenesis process and therefore appear
to be "foreign" to the body. This can include normal proteins that
well evade the immune system, proteins that are usually produced in
very small amount, proteins that are usually only produced at
specific developmental stages, or proteins whose structure is
modified due to mutations.
[0031] Many tumor antigens are known in the art, and many new tumor
antigens can be easily discovered through screening. Non-limiting
examples of tumor antigens include EGFR, Her2, EpCAM, CD20, CD30,
CD33, CD47, CD52, CD 133, CEA, gpA33, mucin, TAG-72, CIX PSMA,
folate binding protein, GD2, GD3, GM2, VEGF, VEGFR, Integrin,
.alpha.V.beta.3, .alpha.5.beta.1, ERBB2, ERBB3, MET, IGF1R, EPHA3,
TRAILR1, TRAILR2, RANKL, FAP and Tenascin.
[0032] In some aspects, Fab or VHH fragments have specificity for
proteins that are overexpressed on tumor cells compared to
corresponding non-tumor cells. "Corresponding non-tumor cells"
refers to non-tumor cells that have the same cell type as the cells
from which the tumor cells are derived. It should be noted that
such proteins are not necessarily different from tumor antigens.
Non-limiting examples include carcinoembryonic antigen (CEA), which
is overexpressed in most colon, rectal, breast, lung, pancreatic,
and gastrointestinal cancers; mygulin receptor (HER-2, neu or
c-erbB-2), which is usually overexpressed in breast, ovarian,
rectal, lung, prostate and cervical cancers; epidermal growth
factor receptor, which is overexpressed in a series of solid tumors
(including breast cancer, head and neck cancer, non-small cell lung
cancer and prostate cancer); asialoglycoprotein receptor;
transferin receptor; serine protease inhibitor enzyme complex
receptor expressed on liver cells; fibroblast growth factor
receptor (FGFR) over-expressed on pancreatic ductal adenocarcinoma
cells; vascular endothelial growth factor receptor (VEGFR) for
anti-angiogenesis gene therapy; folate receptor selectively
overexpressed in 90% of non-mucinous ovarian cancer;
polysaccharide-protein complexes (glycocalyx) on cell surface;
carbohydrate receptors; and polyimmunoglobulin receptors, which are
beneficial for gene transfer to respiratory epithelial cells and
are attractive for the treatment of lung diseases (such as cystic
fibrosis).
[0033] In some aspects, the Fab portion includes: one or two amino
acid sequences selected from SEQ ID NO: 14-25 (Table 2), or
optionally with one or two or three insertions, deletions or
substitutions.
TABLE-US-00002 TABLE 2 Examples of Fab sequences 1. anti-CD19Fab
light chain-nucleic acid (SEQ ID NO: 14)
GATATTCAGATGACTCAGAGCCCTTCCTCACTGTCCGCCAGCGTCGGGGATCGGGTCACT
ATTACATGCAAAGCAAGCCAGAGCGTCGACTATGATGGGGACTCCTATCTGAACTGGTAC
CAGCAGAAGCCAGGCAAAGCTCCCAAGCGGCTGATCTACGACGCATCAAATCTGGTGAGC
GGCGTCCCTTCCAGATTCTCTGGGAGTGGATCAGGCACAGATTTTACCCTGACAATTAGC
TCCCTGCAGCCTGAGGACTTCGCCACTTACTATTGCCAGCAGAGCACAGAAGATCCATGG
ACTTTTGGCGGGGGAACCAAAGTGGAGATCAAGAGA (VL)
ACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGA
ACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGG
AAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGC
AAGGACAGCACCTACAGCCTCAGCAGCACCCGACGGCTGAGCAAAGCAGACTACGAGAAA
CACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGC
TTCAACAGGGGAGAGTGT (CL) 2. anti-CD19Fab light chain - amino acid
(SEQ ID NO: 15)
DIQMTQSPSSLSASVGDRVTITCKASQSVDYDGDSYLNWYQQKPGKAPKRLIYDASNLVS
GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSTEDPWTFGGGTKVEIKR (VL)
TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDS
KDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (CL) 3. anti-CD19Fab
heavy chain-nucleic acid (SEQ ID NO: 16)
CAGGTGCAGCTGGTCCAGTCCGGGGCAGAAGTGAAGAAACCAGGAGCCTCTGTGAAAGTC
AGTTGTAAGGCTTCAGGCTATACCTTCATCTCTTACTGGATGAACTGGGTGAGACAGGCA
CCAGGACAGGGACTGGAGTGGATGGGACAGATTTGGCCTGGCGATGGGGACACCAACTAT
AATCAGCTGCTGAAGGATAAAGCAACTCTGACCGCCGACAAGAGCGCCTCCACCGCTTAC
ATGGAGCTGTCTAGTCTGCGAAGCGAAGACACAGCTGTGTACTATTGCGCACGGAGAGAA
ACCACAACTGTGGGCCGCTACTATTACGCAATGGATTACTGGGGACAGGGCACACTGGTG
ACTGTCTCAAGC (VH)
GCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGG
GGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCG
TGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCA
GGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACC
TACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTT (CH) 4.
anti-CD19Fab heavy chain-amino acid (SEQ ID NO: 17)
QVQLVQSGAEVKKPGASVKVSCKASGYTFISYWMNWVRQAPGQGLEWMGQIWPGDGDTNY
NQLLKDKATLTADKSASTAYMELSSLRSEDTAVYYCARPETTTVGRYYYAMDYWGQGTLV TVSS
(VH) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS
GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV (CH) 5. anti-CD20Fab light
chain-nucleic acid (SEQ ID NO: 18)
CAGATCGTGCTGAGCCAGAGCCCCGCCATCCTGAGCGCCAGCCCCGGCGAGAAGGTGACC
ATGACCTGCAGGGCCAGCAGCAGCGTGAGCTACATCCACTGGTTCCAGCAGAAGCCCGGC
AGCAGCCCCAAGCCCTGGATCTACGCCACCAGCAACCTGGCCAGCGGCGTGCCCGTGAGG
TTCAGCGGCAGCGGCAGCGGCACCAGCTACAGCCTGACCATCAGCAGGGTGGAGGCCGAG
GACGCCGCCACCTACTACTGCCAGCAGTGGACCAGCAACCCCCCCACCTTCGGCGGCGGC
ACCAAGCTGGAGATCAAGAGG (VL)
ACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGA
ACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGG
AAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGC
AAGGACAGCACCTACAGCCTCAGCAGCACCCTGAGGCTGAGCAAAGCAGACTACGAGAAA
CACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGC
TTCAACAGGGGAGAGTGT (CL) 6. anti-CD20Fab light chain-amino acid (SEQ
ID NO: 19)
QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVR
FSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIKR (VL) TVAAPSVFIF
PPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQGNSQESVTEQDS
KDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (CL) 7. anti-CD20Fab
heavy chain-nucleic acid (SEQ ID NO: 20)
CAGGTGCAGCTGCAGCAGCCCGGCGCCGAGCTGGTGAAGCCCGGCGCCAGCGTGAAGATG
AGCTGCAAGGCCAGCGGCTACACCTTCACCAGCTACAACATGCACTGGGTGAAGCAGACC
CCCGGCAGGGGCCTGGAGTGGATCGGCGCCATCTACCCCGGCAACGGCGACACCAGCTAC
AACCAGAAGTTCAAGGGCAAGGCCACCCTGACCGCCGACAAGAGCAGCAGCACCGCCTAC
ATGCAGCTGAGCAGCCTGAGCAGCGAGGACAGCGCCGTGTACTACTGCGCCAGGAGCACC
TACTACGGCGGCGACTGGTACTTCAACGTGTGGGGCGCCGGCACCACCGTGACCGTGAGC (VH)
GCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGG
GGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCG
TGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCA
GGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACC
TACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTT (CH) 8.
anti-CD20Fab heavy chain-amino acid (SEQ ID NO: 21)
QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSY
NQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVS (VH)
ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS
GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKKV (CH) 9. anti-Her2Fab
light chain-nucleic acid (SEQ ID NO: 2) GATATTCAGA TGACCCAGAG
CCCGAGCAGC CTGTCCGCAA GCGTCGGCGA CCGCGTCACG ATTACCTGCC GTGCAAGCCA
GGATGTCAAC ACCGCAGTGG CTTGGTATCA GCAAAAACCG GGTAAAGCGC CGAAACTGCT
GATTTATAGT GCCTCCTTTC TGTACTCTGG CGTGCCGAGT CGTTTCTCAG GTTCGCGCAG
CGGCACCGAT TTTACCCTGA CGATCAGCTC TCTGCAGCCG GAAGACTTCG CCACGTATTA
CTGCCAGCAA CATTATACCA CGCCGCCGAC CTTTGGTCAA GGCACGAAAG TCGAAATTAA A
(VL) ACGGTGGCTG CACCATCTGT CTTCATCTTC CCGCCATCTG ATGAGCAGTT
GAAATCTGGA ACTGCCTCTG TTGTGTGCCT GCTGAATAAC TTCTATCCCA GAGAGGCCAA
AGTAGAGTGG AAGGTGGATA ACGCCCTCCA ATCGGGIAAC TCCCAGGAGA GTGTCACAGA
GCAGGACAGC AAGGACAGCA CCTACAGCCT CAGCAGCACC CTGACGCTGA GCAAAGCAGA
CTACGAGAAA CACAAAGTCT ACGCCTGCGA AGTCACCCAT CAGGGCCTGA GCTCGCCCGT
CACAAAGAGC TTCAACAGGG GAGAGTGT (CL) 10. anti-Her2Fab light
chain-amino acid (SEQ ID NO: 23)
DIQMTQSPSSLSASVGDPVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPS
RFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTEGQGTKVEIK (VL) TVAAPSVFIF
PPSDEQLKSG TASVVCLLNN FYPREAKVQW KVDNALQSGN SQESVTEQDS KDSTYSLSST
LTLSKADYEK HKVYACEVTH QGLSSPVTKS FNRGEC (CL) 11. anti-Her2Fab heavy
chain-nucleic acid (SEQ ID NO: 24) GAAGTGCAGC TGGTTGAATC TGGCGGTGGC
CTGGTTCAAC CGGGTGGCTC CCTGCGTCTG TCATGTGCAG CATCGGGTTT CAACATTAAA
GATACCTATA TCCACTGGGT TCGTCAGGCA CCGGGTAAAG GCCTGGAATG GGTCGCTCGC
ATTTACCCGA CCAATGGTTA TACGCGTTAC GCGGATAGCG TGAAAGGCCG CTTCACCATC
AGCGCAGACA CCTCTAAAAA CACGGCTTAT CTGCAGATGA ACTCGCTGCG TGCGGAAGAT
ACGGCCGTTT ATTACTGTAG CCGCTGGGGT GGCGACGGCT TTTACGCAAT GGATTACTGG
GGTCAAGGCA CGCTGGTCAC GGTCTCA (VH) GCTAGCACCA AGGGCCCATC GGTCTTCCCC
CTGGCACCCT CCTCCAAGAG CACCTCTGGG GGCACAGCGG CCCTGGGCTG CCTGGTCAAG
GACTACTTCC CCGAACCGGT GACGGTGTCG TGGAACTCAG GCGCCCTGAC CAGCGGCGTG
CACACCTTCC CGGCTGTCCT ACAGTCCTCA GGACTCTACT CCCTCAGCAG CGTGGTGACC
GTGCCCTCCA GCAGCTTGGG CACCCAGACC TACATCTGCA ACGTGAATCA CAAGCCCAGC
AACACCAAGG TGGACAAGAA AGTT (CH) 12. anti-Her2Fab heavy chain-amino
acid (SEQ ID NO: 25)
EVQLVESGGGLVQGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRY
ADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVS (VH)
ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS
GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKKV (CH)
[0034] Any of the antibodies or polypeptides described above may
further include additional polypeptides, for example, a signal
peptide that directs the secretion of the encoded polypeptide, an
antibody constant region as described herein, or other heterologous
polypeptides as described herein.
[0035] Those of ordinary skill in the art will understand that the
antibodies disclosed herein can be modified so that they differ in
amino acid sequence from the natural binding polypeptides from
which they are derived. For example, a polypeptide or amino acid
sequence derived from a specific protein may be similar, for
example it has a certain percentage of identity to the starting
sequence, for example, it may have 60%, 70%, 75%, 80%, 85%, 90%,
95%, 98%, or 99% identity with the starting sequence.
[0036] In addition, conservative substitutions, deletions or
insertions of nucleotides or amino acids can be made in the
"non-essential" amino acid region. For example, a polypeptide or
amino acid derived from a specific protein may be the same as the
starting sequence, except for one or more single amino acid
substitutions, insertions, or deletions (e.g., one, two, three,
four, five, six, Seven, eight, nine, ten, fifteen, twenty or more
individual amino acid substitutions, insertions or deletions). In
certain embodiments, compared to the starting sequence, the
polypeptide or amino acid sequence derived from a particular
protein has one to five, one to ten, or one to twenty individual
amino acid substitutions, insertions, or deletions. In other
embodiments, the antigen-binding polypeptides of the present
invention may contain conservative amino acid substitutions.
[0037] In "conservative amino acid substitutions," one amino acid
residue is replaced by an amino acid residue with a similar side
chain. Amino acid residue families with similar side chains have
been defined in the art, including basic side chains (such as
lysine, arginine, histidine), acidic side chains (such as aspartic
acid, glutamic acid), uncharged polar side chains (such as glycine,
asparagine, glutamine, serine, threonine, tyrosine, cysteine),
non-polar side chains (such as alanine, valine, leucine,
isoleucine, proline, phenylalanine, methionine, tryptophan), .beta.
branched side chains (such as threonine, valine, isoleucine) and
aromatic side chains (such as tyrosine, phenylalanine, tryptophan,
histidine). Therefore, non-essential amino acid residues in
immunoglobulin polypeptides are more suitable to be replaced by
other amino acid residues from the same side chain family. In
another embodiment, the amino acid chain may be replaced by a chain
that is structurally similar but differs in the order or component
of the side chain family members.
[0038] The table below provides non-limiting examples of
conservative amino acid substitutions. The similarity score of 0 or
higher in the table indicates conservative substitutions between
two amino acids.
TABLE-US-00003 C G P S A T D E N Q H K R V M I L F Y W W -8 -7 -6
-2 -6 -5 -7 -7 -4 -5 -3 -3 2 -6 -4 -5 -2 0 0 17 Y 0 -5 -5 -3 -3 -3
-4 -4 -2 -4 0 -4 -5 -2 -2 -1 -1 7 10 F -4 -5 -5 -3 -4 -3 -6 -5 -4
-5 -2 -5 -4 -1 0 1 2 9 L -6 -4 -3 -3 -2 -2 -4 -3 -3 -2 -2 -3 -3 2 4
2 6 I -2 -3 -2 -1 -1 0 -2 -2 -2 -2 -2 -2 -2 4 2 5 M -5 -3 -2 -2 -1
-1 -3 -2 0 -1 -2 0 0 2 6 V -2 -1 -1 -1 0 0 -2 -2 -2 -2 -2 -2 -2 4 R
-4 -3 0 0 -2 -1 -1 -1 0 1 2 3 6 K -5 -2 -1 0 -1 0 0 0 1 1 0 5 H -3
-2 0 -1 -1 -1 1 1 2 3 6 Q -5 -1 0 -1 0 -1 2 2 1 4 N -4 0 -1 1 0 0 2
1 2 E -5 0 -1 0 0 0 3 4 D -5 1 -1 0 0 0 4 T -2 0 0 1 1 3 A -2 1 1 1
2 S 0 1 1 1 P -3 -1 6 G -3 5 C 12
[0039] In some embodiments, antibodies can be conjugated to
therapeutic agents, prodrugs, peptides, proteins, enzymes, viruses,
lipids, biological effect modifiers, or pharmaceutical
formulations.
[0040] These antibodies can be conjugated or fused to therapeutic
agents, which may include detectable labels (such as radiolabels),
immunomodulators, hormones, enzymes, oligonucleotides,
photosensitizing therapeutic agents or diagnostic agents, cytotoxic
agents (drugs or toxins), ultrasound enhancers, non-radioactive
labels, their combinations, and other agents known in the art.
[0041] By coupling to a chemiluminescent compound to label the
antibody, the antibody can be detected. Then, by detecting the
presence of fluorescence (which occurs during the chemical
reaction), the presence of chemiluminescent labeled antigen-binding
peptides is determined. Examples of extremely useful
chemiluminescent labeling compounds are luminol, isoluminol,
theromatic acridinium ester, imidazole, acridinium salt and oxalate
ester.
[0042] As mentioned above, the bispecific antibodies, vanants or
derivatives thereof of the present invention can be used in certain
methods of treatment and diagnosis related to cancer or infectious
diseases.
Treatment and Therapy
[0043] The present invention further relates to antibody-based
therapy, which involves administering the bispecific antibody of
the present invention to patients, such as animals, mammals, and
humans, to treat one or more diseases or symptoms described herein.
The therapeutic composition of the present invention includes, but
is not limited to, the antibody of the present invention (including
the variants and derivatives thereof described herein) and nucleic
acid or polynucleotide (including the variants and derivatives
thereof described herein) encoding the antibody of the present
invention.
[0044] The antibodies of the present invention can be used to
treat, inhibit or prevent the following diseases, disorders, or
conditions, including, for example, malignant diseases, disorders,
or conditions (such as cancers) associated with increasing cell
survival or inhibiting cell apoptosis. The cancers include but are
not limited to follicular lymphoma, cancers with p53 mutations, and
hormone-dependent tumors (including but not limited to colon
cancer, heart tumors, pancreatic cancer, melanoma, retinoblastoma,
malignant glioma, lung cancer, colorectal cancer, testicular
cancer, gastric cancer, neuroblastoma, myxoma, myoma, lymphoma,
endothelioma, osteoblastoma, osteoclastoma, osteosarcoma,
chondrosarcoma, adenocarcinoma, breast cancer, prostate cancer,
Kaposi's sarcoma); autoimmune disorders (such as multiple
sclerosis, Sjogren syndrome. Grave disease. Hashimoto's
thyroiditis, autoimmune diabetes, biliary cirrhosis, Behcet's
disease, Crohn's disease, polymvositis, systemic lupus
erythematosus, immune-related glomerulonephritis, autoimmune
gastritis, autoimmune thrombocytopenic purpura and rheumatoid
arthritis); and viral infections (such as herpes virus, poxvirus
and adenovirus), inflammation, graf-versus-host disease (acute
and/or chronic), acute graft rejection, and chronic graft
rejection. The antigen-binding polypeptides, variants or
derivatives thereof of the present invention are used to inhibit
the development, evolution and/or metastasis of cancer, especially
the cancers listed in the above or subsequent paragraphs.
[0045] The antibody of the present invention can also be used to
treat infectious diseases caused by microorganisms or kill
microorganisms by targeting microorganisms and immune cells to
affect the elimination of microorganisms. In one aspect, the
microorganism is a virus (including RNA and DNA viruses),
gram-positive bacteria, gram-negative bacteria, protozoa, or
fungi.
[0046] The specific dosage and treatment regimen for any particular
patient will depend on many factors, including specific
antigen-binding polypeptide, its variants or derivatives used,
patient's age, weight, general health, gender, diet, time of
administration, excretion rate, combination of drugs, and severity
of the specific disease being treated. The physician's judgment on
such factors falls within the judgment of those of ordinary skill
in the art. The dosage will also be based on the individual patient
being treated, route of administration, type of formulation, the
characteristics of the composition used, the severity of the
disease, and the desired effect. The dosage can be determined by
the principles of pharmacology and pharmacokinetics well known in
the art.
[0047] Methods of administration of the bispecific antibodies and
variants include, but are not limited to, intradermal,
intramuscular, intraperitoneal, intravenous, subcutaneous,
intranasal, epidural and oral routes. The antigen-binding
polypeptide or composition can be administered by any convenient
route, for example, by infusion or bolus injection, or absorption
through epithelial or mucosal protective layer (such as oral
mucosa, rectum and intestinal mucosa, etc.); it can be used in
combination with other biologically active preparations. Therefore,
the pharmaceutical composition containing the antigen-binding
polypeptide of the present invention can be administered via oral
cavity, rectum, parenteral, vaginal, abdominal cavity, topically
(such as by powder, ointment, drops or skin patch), buccal, or oral
or nasal cavity.
[0048] The term "parenteral" as used herein refers to the mode of
administration, which includes intravenous, intramuscular,
intraperitoneal, intrasternal, subcutaneous and intraarticular
injections and infusions.
[0049] Administration can be systemic or local. In addition, it is
expected that the antibody of the present invention can be
introduced into the central nervous system by any suitable route,
including intracerebroventricular and intrathecal injection;
intraventricular catheters, for example, connected to a reservoir
(such as Ommaya reservoir), can be used to facilitate
intraventricular injection. Pulmonary administration can also be
used, for example by using inhalers or nebulizers and formulations
with aerosols.
[0050] It may be desirable to apply the bispecific antibody or
composition of the present invention locally to the area in need of
treatment; this can be achieved, for example, but not limited to,
local infusion during surgery, local application (for example,
combined use with wound dressings), injections, catheters,
suppositories, or implants (implants are porous, non-porous,
non-permeable or gel-like materials, including films (such as
silicone membranes) or fibers)). Preferably, when administering a
protein (including an antibody) including the antibody of the
present invention, care should be taken to use a material that does
not adsorb the protein.
[0051] The effective amount of the antibody of the present
invention in the treatment, inhibition and prevention of
inflammation, immune or malignant diseases, disorders or conditions
can be determined by standard clinical techniques. In addition, in
vitro assays can optionally be used to help identify the optimal
dose range. The exact dosage used in the formulation will also
depend on the route of administration and the severity of the
disease, disorder, or condition, and should be determined based on
the judgment of the practitioner and the circumstances of each
patient. The effective dose can be deduced from a dose-response
curve derived from an in vitro or animal model test system.
[0052] As a general suggestion, the dose of the antigen-binding
polypeptide of the present invention administered to a patient is
usually 0.1 mg/kg to 100 mg/kg patient body weight, 0.1 mg/kg to 20
mg/kg patient body weight, or 1 mg/kg to 10 mg/kg patient body
weight. In general, due to the immune response to foreign
polypeptides, human antibodies have a longer half-life in the human
body than antibodies from other species. Therefore, lower dosing
dosage and lower dosing frequency of human antibodies are usually
possible. Moreover, the administration frequency and dosage of the
antibodies of the present invention can be reduced by modification
(such as lipidation) to enhance the uptake of these antibodies and
tissue penetration (such as, into the brain).
[0053] Methods for the treatment of infections or malignant
diseases, disorders or conditions (including administering the
antibodies, variants or derivatives of the present invention),
before being used in humans, are usually tested in vitro, and then
in vivo in acceptable animal models to obtain the desired
therapeutic or preventive activity. Suitable animal models
(including transgenic animals) are well known to those of ordinary
skill in the art. For example, in vitro experiments that
demonstrate the therapeutic utility of the antigen-binding
polypeptides described herein include the effect assays of the
antigen-binding polypeptides on cell lines or patient tissue
samples. The effects of the antigen-binding polypeptide on cell
lines and/or tissue samples can be determined using techniques
known to those skilled in the art, such as tests disclosed
elsewhere herein. According to the present invention, in vitro
tests that can be used to determine whether a specific
antigen-binding polypeptide needs to be used include in vitro cell
culture tests in which a patient tissue sample is grown in a
culture medium and exposed to or otherwise administered with
antibodies and observing the effect of this antibody on the tissue
sample.
[0054] In a further embodiment, the composition of the present
invention is administered in combination with an antitumor agent,
antiviral agent, antibacterial agent or antibiotic preparation or
antifungal preparation. Any of these formulations known in the art
can be administered in the composition of the present
invention.
[0055] In another embodiment, the composition of the invention is
administered in combination with a chemotherapeutic agent. The
chemotherapeutic agents that can be administered with the
composition of the present invention include, but are not limited
to, antibiotic derivatives, such as doxorubicin, bleomycin,
daunorubicin, defensin; anti-estrogens such as tamoxifen;
antimetabolites such as fluorouracil, 5-FU, methotrexate,
fluorouridine, interferon .alpha.-2b, glutamic acid, pracamycin,
mercaptopurine and 6-mercaptoguanine; cytotoxic agents such as
Carmustine, BCNU, lomustine, CCNU, cytarabine, cyclophosphamide,
estramustine, hydroxyurea, procarbazine, mitomycin, busulfan,
cisplatin and vincristine sulfate; hormones such as
medroxyprogesterone, estramustine sodium phosphate, ethinyl
estradiol, estradiol, megestrol acetate, medroxyprogesterone,
diethylstilbestrol diphosphate, chlorinestrol, and testosterone;
nitrogen mustard derivatives, such as chlorambucil, chlorambucil,
dichloromethyldiethylamine (chlorambucil), thiotepa; steroids such
as betamethasone sodium phosphate; and others, such as dacarbazine,
asparaginase, mitotane, vincristine sulfide, vinblastine sulfide,
and etoposide.
[0056] In another embodiment, the composition of the invention is
administered in combination with cytokines. Cytokines that can be
administered with the composition of the present invention include
but are not limited to IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-10,
IL-12, IL-13, IL-15, anti-CD40, CD40L and TNF-.alpha..
[0057] In other embodiments, the composition of the invention is
administered in combination with other therapeutic or preventive
therapies (such as radiation therapy).
Pharmaceutical Compositions
[0058] The invention also provides a pharmaceutical composition.
Such a composition includes an effective amount of the antibody and
an acceptable carrier. In a specific embodiment, the term
"pharmaceutically acceptable" means approved by a regulatory agency
of the federal or state government, or listed in the U.S.
Pharmacopoeia or other generally recognized pharmacopoeia for use
in animals, more specifically for human. Further, the
"pharmaceutically acceptable carrier" will usually be a non-toxic
solid, semi-solid or liquid filler, diluent, encapsulating material
or any type of excipient.
[0059] The term "carrier" refers to a diluent, adjuvant, excipient
or carrier with which the drug is used. Such pharmaceutical
carriers can be sterile liquids, such as water and oils, including
oils of petroleum, animal, vegetable, or synthetic origin, such as
peanut oil, soybean oil, mineral oil, sesame oil, and the like.
When the pharmaceutical composition is administered intravenously,
water is the preferred carrier. Salt solutions and aqueous glucose
and glycerin solutions can also be used as liquid carriers,
especially for injectable solutions. Suitable pharmaceutical
excipients include starch, glucose, lactose, sucrose, gelatin,
malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl
monostearate, talc, sodium chloride, skimmed milk powder, glycerol,
propylene, ethylene glycol, water, ethanol, etc.
[0060] If desired, the composition may also contain small amounts
of wetting or emulsifying agents or pH buffering agents, such as
acetate, citrate or phosphate. It can also be expected to add
antibacterial agents such as benzyl alcohol or methyl benzoate,
antioxidants such as ascorbic acid or sodium bisulfite, chelating
agents such as ethylenediaminetetraacetic acid and agents for
isotonicity adjustment such as chlorine sodium or dextrose. These
compositions can take the form of solutions, suspensions, tablets,
pills, capsules, powders, sustained-release formulations and the
like. The composition can be formulated as a suppository using
customary binders and carriers such as triglycerides. Oral
formulations can include standard carriers such as pharmaceutical
grades of mannitol, lactose, starch, magnesium stearate, sodium
saccharin, cellulose, magnesium carbonate, and the like. Examples
of suitable pharmaceutical carriers are described by E. W. Martin
in Remington's Pharmaceutical Sciences, incorporated by reference.
Such a composition will contain a therapeutically effective amount
of the antigen-binding polypeptide (preferably in a purified form)
and an appropriate amount of carrier so as to provide the patient
with an appropriate mode of administration. This formulation should
suit the mode of administration. This parental preparation can be
contained in ampoules, disposable syringes or multiple dose vials
made of glass or plastic.
[0061] In one embodiment, the composition is formulated into a
pharmaceutical composition suitable for intravenous administration
to humans according to routine procedures. Generally, the
composition for intravenous administration is a solution in a
sterile isotonic aqueous buffer. If necessary, the composition may
also include a solubilizer and a local anesthetic such as lidocaine
to relieve pain at the injection site. The components are usually
supplied individually or mixed together in unit dosage form, for
example, a lyophilized powder or an anhydrous concentrate in a
closed container, for example, an ampoule or sachette indicating
the number of active agents. When the composition is administered
by infusion, it can be dispersed in an infusion bottle containing
sterile pharmaceutical grade water or saline. When the composition
is administered by injection, an ampoule of sterile water or saline
for injection may be provided so that the ingredients can be mixed
before administration.
[0062] The composition of the present invention can be formulated
in a neutral or salt form. Pharmaceutically acceptable salts
include salts formed from anions, such as those derived from
hydrochloric acid, phosphoric acid, acetic acid, oxalic acid,
tartaric acid, etc., and salts formed from cations, such as those
derived from sodium, potassium, ammonium, calcium, hydrogen Salts
of iron oxide, isopropylamine, triethylamine,
ethylhydroxyethylamine, histidine, procaine, etc.
Examples
S-Fab Design and Protein Purification
[0063] The structure of S-Fab is shown in FIG. 1A. The VH-CH1 and
VL-CL fragments of anti-CD3 were synthesized and cloned by standard
DNA cloning techniques. A signal sequence pelB was added to the
N-terminus for periplasmic expression. For site-specific
conjugation of PEG, a cysteine residue was added at the C-terminal
of light chain followed by a short linker, and then another
cysteine (CGGGGC) and a his6 tag. S-Fab was constructed via the
heterodimerization of VL-CL/VH-CH1 (anti-CD3 Fab) with anti-CEA VHH
nanobody. Flag-tag was added to the C-terminal of heavy chain for
detection (FIG. 1B).
[0064] To produce the S-Fab, the two plasmids encoding respective
VH--CH--VHH and VL-CL polypeptides were co-transformed into BL21
(DE3, codon plus) competent cells with proper antibiotics. When the
absorbance (OD600) of cell culture reached 0.8, 0.2 mM
isopropyl-.beta.-d-thiogalactoside (IPTG) was added to induce
protein expression. The cells were cultured at 16.degree. C. for
another 40 hrs before harvesting. After harvesting the cells by
centrifugation, periplasmic extraction was performed by
re-suspending the cell pellets 1:4 (g:mL) in a pre-cooled sucrose
solution (20 mM Tris-HCl pH 7.5; 25% (w/v) sucrose; 1 mM EDTA).
After 15 min incubation on ice, the suspension was centrifuged at
10,000 g for 20 min, and the supernatant fraction was collected as
the sucrose fraction. The pellet was then re-suspended in a chilled
periplasmic solution (5 mM MgCb) and centrifuged at 10,000 g for 20
min. The supernatant was gathered as the periplasmic fraction.
[0065] The S-Fab protein was then purified from the combined
sucrose and periplasmic fractions by a two-step purification: first
by the immobilized Ni-NTA affinity chromatography (GE Health, USA),
and then by an IgG-CH1 affinity matrix (Lot, 194320005; hermoFisher
Scientific Inc, USA) (FIG. 1C). Gel filtration analysis was
performed using a Bio-Rad FPLP system and GE Superdex 200.TM.
Increase 10/300 GL column with a flow rate of 0.5 ml/min. Fractions
(0.5 ml per fraction) were collected, and then subjected to the
SDS-PAGE analysis under reducing conditions. The resulting
fractions were visualized by the coomassie blue staining. Protein
markers (Lot, MWGF200; Sigma Aldrich Co.. Ltd. USA) were loaded as
standard controls for gel filtration analysis. Gel filtration
analysis showed the intact S-Fab antibodies with molecular weights
of .about.130 kD (FIG. 1D), indicated the dimerization of S-Fab
(.about.65 kD of monomer), likely formed by the disulfide bond(s)
of C-terminal cysteine residues of VL-CL.
Conjugation of S-Fab to PEG (PEGylation)
[0066] The S-Fab was engineered with two terminal cysteine residues
located at the C-terminal of CL, which served as the sites for
conjugation with a 20 kDa linear MAL-PEG-OMe. S-Fab (approximately
1.35 mg/mL (about 20 .mu.M) in 5.0 mL phosphate buffered saline
(PBS, pH 7.4) and three molar equivalents of 1 mM tris
(2-carboxyethyl) phosphine (TCEP, final 60 .mu.M, approximately 300
.mu.L) were mixed and incubated for two hours at 22.degree. C. to
obtain the reduced S-Fab fragments.
[0067] In order to explore the optimal molar ratio of MAL-PEG-OMe
and S-Fab in the PEGylation process, we proceeded a series of
reactions with the molar equivalent of PEG:S-Fab as 0:1, 10:1,
20:1, 40:1 and 60:1, respectively (FIG. 2A). MAL-PEG-OMe was
dissolved in the sterile water to obtain a working concentration of
20 mg/mL (1 mM). The PEGylation of S-Fab was carried out by mixing
the MAL-PEG-OMe (at the working concentration) with reduced S-Fab
and shaking at 22.degree. C. for 2 hrs. The resulting samples were
subjected to 12% SDS-PAGE electrophoresis (5 ul/sample/PAGE), then
for coomassie blue and barium iodide staining of PEG according to
reference (FIGS. 2A and 2B). After electrophoresis, the western
blot assay was used to detect the PEGylated chain. Briefly, another
two gels were transferred to polyvinylidene fluoride membrane
(Millipore, USA). After two-hour blocking with 5% skimmed milk,
membranes were respectively incubated with mouse monoclonal
anti-flag HRP (1:2000, for heavy chain) and mouse monoclonal
anti-his IgG (1:3000, for light chain) in 5% skimmed milk. The
secondary antibody (goat anti-mouse HRP-conjugated IgG, 1:3000)
were incubated with the light chain membrane for another one hour
after washing with TBST buffer. The membranes were developed with
Pierce's West Pico chemiluminescence substrate (Millipore, USA)
after washing with TBST buffer. PEGylated S-Fab was purified using
an AKTA avant25 FPLC purification system (GE Healthware, USA) and
Superdex 10/300 GL column with a flow rate of 0.8 ml/min. The
column was first equilibrated with two column volumes (CV) of
distilled water and then two CV of PBS before applying samples. All
the collected fractions were analyzed by coomassie blue and barium
iodide complex dye after SDS-PAGE at reducing conditions. Fractions
for the purified PEGylated S-Fab were pooled together for further
studies.
[0068] Increased ratio of PEG:S-Fab in the PEGylation of S-Fab with
MAL-PEG-OMe resulted in a higher molecular weight band (.about.107
kD and .about.45 kD), indicating the maleimide functionalized PEG
was conjugated to S-Fab. The 45 kD band suggested the single
conjugation at the single C-terminal cysteine (FIGS. 2B and 2D).
The .about.107 kD and indicates both cysteine residues at the VL-CL
terminal were PEGylated (FIGS. 2B and 2D). The .about.107 kD band
appeared first and accounts for the majority of light chain,
indicating the preferred conjugation on both cysteine residues
(FIGS. 2B and 2D). As no decrease of VH--CH-VHH was observed (FIGS.
2A and 2C) and no high molecular weight of VH--CH1-VHH was observed
based on western blot (FIG. 2C), suggesting no PEGylation on the
VH--CH1-VHH chain. As the band of VL-CL chain was decreased (FIGS.
2A and 2D), and PEGylation corresponds to the VL-CL chain based on
anti-his western blot (FIGS. 2B and 2D), it suggested only VL-CL
had PEGylation. As the molar ratio of PEG:S-Fab increases, there
are also slightly increased higher molecular weight PEGylated
bands, suggesting further PEGylation on the other cysteine residues
on VH-CH1 or VL-CL. The molar ratio of PEG:S-Fab at 20 was then
chosen for conjugation as it had higher VL-CL conjugation without
higher molecular weight conjugation.
[0069] To remove the free PEG, free S-Fab, and high molecular
weight proteins, the conjugation reaction mixture was subjected to
size exclusion analysis. Based on SDS-PAGE followed by coomassie
blue staining (FIG. 3B) and barium iodide staining for PEGylation
(FIG. 3C), the high molecular weight fraction 1 (FIG. 3A-3C)
contained a large proportion of multiple conjugation species.
Fraction 2 (FIG. 3A-3C) contains mostly the double and single site
conjugated PEG-S-Fab, while fraction 3 (FIG. 3A-3C) containing free
S-Fab, free PEG, and single site conjugated S-Fab. Fractions
containing mostly the double and single site conjugated PEG-S-Fab
(Fraction 2 in FIG. 3) were pooled for further studies.
PEG-S-Fab can bind the tumor antigen CEA and CD3.sup.+ T cells.
[0070] The bispecific S-Fab has two different binding sites, the
anti-CEA VHH recognizing CEA on tumor cells, and the anti-CD3
recognizing CD3' on T cells. To check whether PEGylation of S-Fab
affects the binding of CEA-positive cancer cells, flow cytometry
analysis was performed using LS174T cells, which have
over-expression of CEA. CEA-negative cell line SKOV3 was used as
negative control.
[0071] Briefly, 1.times.106 (for LS174T and SKOV3) or 5.times.105
(for T cells) cells per sample were collected by centrifugation at
1000 rpm for 5 min and then washed once with 1.0 mL of ice-cold PBS
with 0.2% bovine serum albumin (BSA). The primary antibodies,
including S-Fab, PEG-S-Fab, and the blank control (Vehicle, PBS
only) were added to a final concentration of 10 .mu.g/mL, and then
incubated on ice for one hour followed by washing twice with
ice-cold PBS with 0.1% BSA. Anti-CD3 FITC (OKT3, final
concentration of 10 .mu.g/mL) was used as positive control for
CD3.sup.+ antigen binding analysis. Goat anti-human IgG
(H+L)-AlexaFluor 488 antibody was then added to a final
concentration of 5 .mu.g/mL. The cells were incubated on ice for
another hour. After washing the cells twice, flow cytometric
detection was then performed.
[0072] Both S-Fab and PEG-S-Fab showed obviously specific
fluorescence intensity shifts, suggesting that PEG-S-Fab can still
bind to LS174T cells (FIG. 4A). The slight left shift of PEG-S-Fab
to the value of S-Fab (FIG. 4B) indicates that the PEGlyation of
S-Fab may slightly reduce the binding affinity of S-Fab to its
antigen. For SKOV3 cells, both S-Fab and PEG-S-Fab cannot bind the
cells due to the lack of CEA antigen on cell surface, suggesting
that PEG-S-Fab retains the specific antigen binding (FIG. 4B).
Similarly, for the CD3.sup.+ on the T cells, the obvious
fluorescence intensity shift of S-Fab and PEG-S-Fab was also
presented. PEGylation may also slightly decrease the binding
activity to the CD3.sup.+ antigen on T cells (FIG. 4C). The only
slightly reduced binding activities of PEG-S-Fab suggested that
site specific conjugations in the middle of S-Fab had minimal
effects on bispecific antibody binding affinity.
[0073] To further analyze the binding of S-Fab and PEG-S-Fab to
cell surface CEA, immunofluorescence assay was performed. Briefly.
LS174T and SKOV3 cells (2.5.times.105 cells in 1.0 mL,
respectively) were plated on 30-mm confocal glass bottom dishes
(NEST, cat. 801002) to 80% confluence. The cells were then washed
with cold PBS three times before fixing with 4% paraformaldehyde.
The fixed cells were incubated with 20 .mu.g of S-Fab or PEG-S-Fab
and then 10 .mu.g goat anti-human IgG (H+L)-AlexaFluor 488 antibody
for 2 hour at 4.degree. C. respectively. The cell nuclei were
counterstained with DAPI. After washing with PBS, samples were then
examined using Olympus FV3000 laser scanning confocal microscope
and analyzed by Olympus FV31S-SW_V2.1 software.
[0074] Both S-Fab (upper panel in FIG. 4D) and PEG-S-Fab (upper
panel in FIG. 4E) can bind CEA positive LS174T cells. For
CEA-positive SKOV3 cells, no binding was observed for S-Fab (lower
panel in FIG. 4D) or PEG-S-Fab (lower panel in FIG. 4E), further
supporting that PEGlyation maintains the specific binding affinity
of S-Fab to its antigen.
PEG-S-Fab has Potent Specific Cytotoxicity Against Tumor Cells
[0075] The CEA-positive human LS174T cells and the CEA-negative
human SKOV3 cells were used to assess the in-vitro growth
inhibitory effects of S-Fab and PEG-S-Fab. Briefly, LS174T and
SKOV3 cells were used as target cells (T), and freshly prepared
human CD3.sup.+ T cells without prior stimulation were used as
effector cells (E). In vitro cytotoxicity assays were performed in
96-well microplates in triplicates by seeding 5,000 target cells
per well in 100 .mu.L of corresponding media. After a 6-hour
incubation, an equal volume of CD3.sup.+ T cells were added to each
well at an E:T ratio of 10:1, and a series of concentrations
(0.033, 0.1, 0.33, 1, 3.3, 10, 33 and 100 nM) of S-Fab or PEG-S-Fab
were then added correspondingly. After 72-hour incubation, cell
viability was evaluated via a CCK8 assay according to the
manufacturer protocol. The absorbance values were detected using a
TECAN microplate reader at 450 nm. The survival rate (100%) was
calculated as: [(As-Ab)(A0-Ab)].times.100%, where As is the
absorbent value of the measurement group, Ab is the absorbent value
of medium and A0 is the absorbent value of measurement group at 0
nM.
[0076] Both S-Fab and PEG-S-Fab can kill CEA-positive LS174T cells
efficiently even at 0.033 nM (FIG. 5A) while no cytotoxcity against
CEA-negative SKOV3 cells (FIG. 5B). The PEG-S-Fab has slightly
decreased cytotoxicity than S-Fab (FIG. 5A). This could be due to
the slightly decreased antigen binding or steric interference of
PEGylation. However, the level of decreased activity seems much
lower than many other PEGlyated proteins.
PEGylation Prolongs In Vivo Half-Life of S-Fab
[0077] To determine the circulating half-life of PEG-S-Fab, the
intravenous PK profiles of S-Fab and PEG-S-Fab in rat were
analyzed. SPF male SD rats (250-300 g) were used for the PK assay.
Food was controlled to maintain animals below 350 g in weight.
S-Fab (1.0 mg/kg), PEG-S-Fab (1.0 mg/kg) or a volume equivalent of
vehicle solution PBS was administered through the caudal vein. The
blood sample (each approximately 150-200 .mu.L) was taken from the
orbital vein using a capillary under isoflurane anesthesia at 0,
0.5, 1, 2, 4, 8, 16, 24, 36, 48, 72, 96 and 144 hrs after
administration. All blood samples were collected into heparinized
tubes. Plasma was obtained via centrifugation at 3,500 g for 30
min, and then stored at -80.degree. C. until further analysis.
[0078] S-Fab and PEG-S-Fab in the plasma samples were quantified
using an ELISA assay. Briefly, a 100 .mu.L aliquot of 6D6 (mouse
anti-human IgG Fab antibody) (1.0 .mu.g/mL in PBS) was coated to
each well of a 96-well ELISA microplate (ThermoFisher, USA) for 2
hrs at 37.degree. C. The wells were then washed twice with 200
.mu.L of PBST (PBS+0.05% Tween-20). Wells were then blocked with
200 .mu.L of blocking buffer (PBST containing 1% bovine serum
albumin) for 2 hrs at 37.degree. C. Each well was then washed five
times with PBST prior to the addition of 100 .mu.L of samples or
standards. Samples and standards (100, 80, 50, 40, 30, 20, 10, 5, 1
and 0.1 .mu.g/mL) were prepared in the blocking buffer with the
standards (S-Fab) prepared in a 1:10 dilution of plasma using PBS,
which was important to avoid matrix effects in the assay. For
plasma samples, a 1:3 dilution was used. 100 .mu.L sample or
standard aliquots was then added in triplicate and incubated at
37.degree. C. for one hour. Each well was washed again with PBST
before the addition of 100 .mu.L of secondary antibody (mouse
monoclonal anti-flag mzperoxidase (HRP) antibody at 1:500
dilutions) per well at 37.degree. C. for one hour. After washing
for five times, a 100 .mu.L aliquot of TMB substrate solution was
added to each well. After 10 min incubation, 100 .mu.L of 2M
H.sub.2SO.sub.4 was added to stop the reaction. The absorbance was
then detected at 450 nm using a TECAN ELISA microplate reader.
Serum elimination t.sub.1/2 and clearance were calculated by 3p97
pharmacokinetic software using standard formula. The results are
expressed as the mean.+-.SEM, and comparisons between the groups
were made with an unpaired Student's t-test. Differences were
considered to be statistically significant if p<0.05.
[0079] S-Fab and PEG-S-Fab were quantified using the ELISA method
as described in the part of 210. Quantification of a series of
standards showed that the standard curve was y=0.0934.times.+0.0748
(0.ltoreq.x.ltoreq.25 .mu.g/mL) with R.sup.2 value of 0.9982 (FIG.
6A). Based on serum protein concentration up to 144 hrs, the S-Fab
had an in-vivo half-life of approximately 3.0 hrs, similar to
reported half-life of other Fab in vivo [34-36]. PEGylation
significantly extended the half-life of PEG-S-Fab to 36.0 hrs, a
12-fold increase to S-Fab (FIG. 6B).
[0080] S-Fab and PEG-S-Fab stability was assessed in human fresh
plasma over two weeks. Briefly. S-Fab and PEG-S-Fab were diluted
with human fresh plasma (without platelet), which generated an
initial concentration of 100 .mu.g/mL. The samples were incubated
at 37.degree. C. for two weeks. At the time intervals of 0, 24, 48,
72, 96, 168, 264 and 336 hr, 40 .mu.L samples were collected and
then stored directly at -80.degree. C. till further analysis. The
samples were thawed on ice and then centrifuged at 14,000 rpm for
10 mins at 4.degree. C. The supernatant was then subjected to
electrophoresis on 12% SDS-PAGE (5 ul sample per well). After
electrophoresis, western blot as descripted in 2.4 was performed to
analyze the protein level. When incubated with human plasma in
vitro at 37.degree. C., the level of S-Fab decreased sharply after
72 hrs (FIGS. 6C and 6E). For PEG-S-Fab, the decrease was much
slower (FIGS. 6D and 6F). Thus. PEGylation can also improve the
stability of S-Fab in plasma probably due to the protection of
enzymatic digestion in plasma.
PEG-S-Fab Induces More Potent In Vivo Anti-Tumor Activity
[0081] The increased in vivo half-life and comparable in vitro
cytotoxicity of PEG-S-Fab prompted us to assess the in vivo
anti-tumor activity of PEG-S-Fab in an adoptive xenograft model.
The in vivo anti-tumor activities of S-Fab and PEG-S-Fab were
studied using NOD/SCID mice engrafted subcutaneously with LS174T
cells. Briefly, LS174T cells were harvested and washed once with
PBS, and then mixed with human PBMCs freshly isolated from healthy
donors. The mixtures of 1.times.10.sup.6 LS174T cells and
5.times.10.sup.6 human PBMCs were injected subcutaneously into the
right flank of NOD/SCID mice in a total volume of 0.2 mL per mouse.
One hour after the engraftment, 0.3 nmol S-Fab (20.0 .mu.g per
mouse), and 0.3 nmol PEG-S-Fab (32.0 .mu.g per mouse) or the
vehicle control (PBS) were injected intraperitoneally. The animals
were then treated daily (0.3 nM per mouse in each group) over the
following six days. Tumor volume was measured with calipers in two
perpendicular dimensions and was calculated using the formula
(width.sup.2.times.length)/2. All data were expressed as the mean
SE for each group, and differences between groups were determined
by two-way ANOVA using GraphPad Prism 5 software.
[0082] When NOD/SCID mice were transplanted with LS174T cells and
fresh isolated human PBMCs, rapid tumor growth was observed.
Compared with the vehicle group, significant tumor growth
inhibition (p<0.01) was observed when the mice were treated with
either S-Fab or PEG-S-Fab in the presence of human PBMCs (FIG. 7).
The tumor growth inhibition was more pronounced in the presence of
PEG-S-Fab compared to S-Fab (p<0.01), indicating PEGylation of
S-Fab protein can enhance the therapeutic efficacy of the S-Fab in
the xenograf mouse model.
[0083] All patents and other references cited in the specification
are indicative of the level of skill of those skilled in the art to
which the invention pertains, and are incorporated by reference in
their entireties, including any tables and figures, to the same
extent as if each reference had been incorporated by reference in
its entirety individually.
[0084] One skilled in the art would readily appreciate that the
present invention is well adapted to obtain the ends and advantages
mentioned, as well as those inherent therein. The methods,
variances, and compositions described herein as presently
representative of preferred embodiments are exemplary and are not
intended as limitations on the scope of the invention. Changes
therein and other uses will occur to those skilled in the art,
which are encompassed within the spirit of the invention, are
defined by the scope of the claims.
[0085] In addition, where features or aspects of the invention are
described in terms of Markush groups or other grouping of
alternatives, those skilled in the art will recognize that the
invention is also thereby described in terms of any individual
member or subgroup of members of the Markush group or other
group.
[0086] Also, unless indicated to the contrary, where various
numerical values are provided for embodiments, additional
embodiments are described by taking any 2 different values as the
endpoints of a range. Such ranges are also within the scope of the
described invention.
[0087] When appropriate, the explanatory description herein may be
implemented in the absence of any one or more elements, one or more
restrictions that are not specifically disclosed herein. Therefore,
for example, in each case herein, any one of the terms "including",
"substantially consisting of" and "consisting of" can be replaced
by the other two terms. Therefore, it should be understood that
although the present invention has been specifically disclosed
through preferred embodiments and optional features, those skilled
in the art can make modifications and changes to the ideas
disclosed herein, and these modifications and changes are still
within the scope of the present invention as defined in the
appended claims.
Sequence CWU 1
1
261120PRTArtificial Sequenceanti-CEA VHH 1Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Phe Val Gln Ala Gly Glu1 5 10 15Ser Leu Thr Leu Ser
Cys Thr Ser Ser Thr Leu Thr Phe Thr Pro Tyr 20 25 30Arg Met Ala Trp
Tyr Arg Gln Ala Pro Gly Lys Gln Arg Asp Leu Val 35 40 45Ala Asp Ile
Ser Ser Gly Asp Gly Arg Thr Thr Asn Tyr Ala Asp Phe 50 55 60Ala Lys
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ile Lys Asn Thr Val65 70 75
80Phe Leu Arg Met Thr Asn Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr
85 90 95Cys Asn Thr Phe Val Ser Phe Val Gly Ile Ala Arg Ser Trp Gly
Gln 100 105 110Gly Thr Gln Val Thr Val Ser Ser 115
1202102PRTArtificial Sequenceanti-CD16 VHH 2Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser
Cys Ser Phe Pro Gly Ser Ile Phe Ser Leu Thr 20 25 30Met Gly Trp Tyr
Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Val Thr 35 40 45Ser Ala Thr
Pro Gly Gly Asp Thr Asn Tyr Ala Asp Phe Val Lys Gly 50 55 60Arg Phe
Thr Ile Ser Arg Asp Asn Ala Arg Ser Ile Ile Tyr Leu Gln65 70 75
80Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Tyr Ala
85 90 95Arg Thr Arg Asn Trp Gly 1003102PRTArtificial
Sequenceanti-CD16 VHH 3Glu Val Gln Leu Val Glu Ser Gly Gly Glu Leu
Val Gln Ala Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Leu Thr Phe Ser Ser Tyr 20 25 30Asn Met Gly Trp Phe Arg Arg Ala Pro
Gly Lys Glu Arg Glu Phe Val 35 40 45Ala Ser Ile Thr Trp Ser Gly Arg
Asp Thr Phe Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser
Arg Asp Asn Ala Lys Asn Thr Val Tyr65 70 75 80Leu Gln Met Ser Ser
Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Ala Asn Pro
Trp Pro 1004102PRTArtificial Sequenceanti-CD16 VHH 4Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Glu1 5 10 15Ser Leu Thr
Leu Ser Cys Val Val Ala Gly Ser Ile Phe Ser Phe Ala 20 25 30Met Ser
Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Val Ala 35 40 45Arg
Ile Gly Ser Asp Asp Arg Val Thr Tyr Ala Asp Ser Val Lys Gly 50 55
60Arg Phe Thr Ile Ser Arg Asp Asn Ile Lys Arg Thr Ala Gly Leu Gln65
70 75 80Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn
Ala 85 90 95Gln Thr Asp Leu Arg Asp 1005102PRTArtificial
Sequenceanti-CD16 VHH 5Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly1 5 10 15Ser Leu Thr Leu Ser Cys Val Ala Ala Gly
Ser Ile Phe Thr Phe Ala 20 25 30Met Ser Trp Tyr Arg Gln Ala Pro Arg
Lys Glu Arg Glu Leu Val Ala 35 40 45Arg Ile Gly Thr Asp Asp Glu Thr
Met Tyr Lys Asp Ser Val Lys Gly 50 55 60Arg Phe Thr Ile Ser Arg Asp
Asn Val Lys Arg Thr Ala Gly Leu Gln65 70 75 80Met Asn Asn Leu Lys
Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn Ala 85 90 95Arg Thr Asp Tyr
Arg Asp 1006123PRTArtificial Sequenceanti-Her2 sdAb 6Gln Val Gln
Leu Val Gln Ser Gly Gly Gly Leu Val Gln Ala Gly Gly1 5 10 15Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Ser Tyr 20 25 30Ala
Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40
45Ala Ala Ile Ser Trp Ser Gly Ala Asn Ile Tyr Val Ala Asp Ser Val
50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asp Thr Val
Tyr65 70 75 80Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95Ala Val Lys Leu Gly Phe Ala Pro Val Glu Glu Arg
Gln Tyr Asp Tyr 100 105 110Trp Gly Gln Gly Thr Gln Val Thr Val Ser
Ser 115 1207128PRTArtificial Sequenceanti-EGFR1 VHH 7Met Ala Glu
Val Gln Gln Ala Ser Gly Gly Gly Leu Val Gln Ala Gly1 5 10 15Gly Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Glu Thr Thr 20 25 30Ser
Ala Ile Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe 35 40
45Val Ala Gln Ile Ser Ala Ser Gly Leu Gly Ile Asn Tyr Ser Gly Thr
50 55 60Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Ala Asp Lys Thr Thr
Val65 70 75 80Tyr Leu Gln Met Asn Ser Leu Thr Pro Glu Asp Thr Ala
Val Tyr Tyr 85 90 95Cys Ala Ala Gly Phe His Tyr Ile Ala Ala Ile Arg
Arg Thr Thr Asp 100 105 110Phe His Phe Trp Gly Pro Gly Thr Leu Val
Thr Val Ser Ser Gly Arg 115 120 1258121PRTArtificial
Sequenceanti-F4+ETEC bacteria VHH 8Gln Val Gln Leu Gln Glu Ser Gly
Gly Gly Leu Val Gln Ala Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Glu
Ala Ser Gly Asn Val Asp Arg Ile Asp 20 25 30Ala Met Gly Trp Phe Arg
Gln Ala Pro Gly Lys Gln Arg Glu Phe Val 35 40 45Gly Tyr Ile Ser Glu
Gly Gly Ile Leu Asn Tyr Gly Asp Phe Val Lys 50 55 60Gly Arg Phe Thr
Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu65 70 75 80Gln Met
Ser Asn Leu Lys Ser Glu Asp Thr Gly Val Tyr Phe Cys Ala 85 90 95Ala
Ser His Trp Gly Thr Leu Leu Ile Lys Gly Ile Glu His Trp Gly 100 105
110Lys Gly Thr Gln Val Thr Val Ser Ser 115 1209127PRTArtificial
Sequenceanti-PS2-8 VHH 9Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Gln Ala Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Arg Ser Phe Ser Arg Asp 20 25 30Ala Met Gly Trp Phe Arg Gln Ala Pro
Gly Lys Glu Arg Asp Val Val 35 40 45Ala Ala Ile Asn Leu Asn Gly Gly
Arg Thr Tyr Ser Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser
Arg Asp Asn Asp Lys Asn Thr Val Tyr65 70 75 80Leu Gln Met Ser Asn
Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Ala Arg Glu
Gly Asp Val Gly Leu Val Ser Tyr Lys Arg Ser Ser 100 105 110Asn Tyr
Pro Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120
12510121PRTArtificial Sequenceanti-Huntavirus VHH 10Met Ala Glu Val
Gln Leu Gln Ala Ser Gly Gly Gly Leu Val Gln Ala1 5 10 15Gly Gly Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Ser Ser 20 25 30Met Tyr
Ser Met Val Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu 35 40 45Phe
Val Ala Gly Ile Ile Trp Thr Ser Ser Leu Thr Tyr Tyr Ala Asp 50 55
60Ser Leu Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr65
70 75 80Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Ile
Tyr 85 90 95Tyr Cys Ala Ala Asp Thr Lys Thr Gly Gly Gly Gly Tyr Glu
Tyr Trp 100 105 110Gly Gln Val Thr Val Thr Val Ser Ser 115
12011119PRTArtificial Sequenceanti-Huntavirus VHH 11Met Ala Glu Val
Gln Leu Gln Ala Ser Gly Gly Gly Leu Val Gln Pro1 5 10 15Gly Gly Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Phe Ser 20 25 30Ser Asp
Val Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu 35 40 45Leu
Val Ala Phe Ile Thr Asp Asp Gly Gly Thr Asn Tyr Ala Asp Ser 50 55
60Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Glu Asn Thr Val65
70 75 80Ser Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr
Tyr 85 90 95Cys Asn Ala Arg Tyr Tyr Ser Gly Gly Tyr Arg Asn Tyr Trp
Gly Gln 100 105 110Val Thr Val Thr Val Ser Ser
11512115PRTArtificial Sequenceanti-CD16 VHH 12Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu
Ser Cys Ser Phe Pro Gly Ser Ile Phe Ser Leu Thr 20 25 30Met Gly Trp
Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Val Thr 35 40 45Ser Ala
Thr Pro Gly Gly Asp Thr Asn Tyr Ala Asp Phe Val Lys Gly 50 55 60Arg
Phe Thr Ile Ser Arg Asp Asn Ala Arg Ser Ile Ile Tyr Leu Gln65 70 75
80Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Tyr Ala
85 90 95Arg Thr Arg Asn Trp Gly Thr Val Trp Gly Gln Gly Thr Gln Val
Thr 100 105 110Val Ser Ser 11513129PRTArtificial Sequenceanti-CD3
VHH 13Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly
Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser
Asn Tyr 20 25 30His Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg
Glu Leu Val 35 40 45Ala Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr
Thr Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asn Asn Ala
Lys Asn Thr Met Ser65 70 75 80Leu Gln Met Ser Asn Leu Lys Pro Glu
Asp Thr Gly Val Tyr Tyr Cys 85 90 95Thr Thr Pro Thr Glu Lys Gly Ser
Ser Ile Asp Tyr Trp Gly Gln Gly 100 105 110Thr Gln Val Thr Val Ser
Ser Gly Arg Tyr Pro Tyr Asp Val Pro Asp 115 120
125Tyr14654DNAArtificial Sequenceanti-CD19Fab light chain-nucleic
acidVL(1)..(336)CL(337)..(654) 14gatattcaga tgactcagag cccttcctca
ctgtccgcca gcgtcgggga tcgggtcact 60attacatgca aagcaagcca gagcgtcgac
tatgatgggg actcctatct gaactggtac 120cagcagaagc caggcaaagc
tcccaagcgg ctgatctacg acgcatcaaa tctggtgagc 180ggcgtccctt
ccagattctc tgggagtgga tcaggcacag attttaccct gacaattagc
240tccctgcagc ctgaggactt cgccacttac tattgccagc agagcacaga
agatccatgg 300acttttggcg ggggaaccaa agtggagatc aagagaacgg
tggctgcacc atctgtcttc 360atcttcccgc catctgatga gcagttgaaa
tctggaactg cctctgttgt gtgcctgctg 420aataacttct atcccagaga
ggccaaagta cagtggaagg tggataacgc cctccaatcg 480ggtaactccc
aggagagtgt cacagagcag gacagcaagg acagcaccta cagcctcagc
540agcaccctga cgctgagcaa agcagactac gagaaacaca aagtctacgc
ctgcgaagtc 600acccatcagg gcctgagctc gcccgtcaca aagagcttca
acaggggaga gtgt 65415218PRTArtificial Sequenceanti-CD19Fab light
chain - amino acidVL(1)..(109)CL(110)..(218) 15Asp Ile Gln Met Thr
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr
Ile Thr Cys Lys Ala Ser Gln Ser Val Asp Tyr Asp 20 25 30Gly Asp Ser
Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro 35 40 45Lys Arg
Leu Ile Tyr Asp Ala Ser Asn Leu Val Ser Gly Val Pro Ser 50 55 60Arg
Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser65 70 75
80Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Thr
85 90 95Glu Asp Pro Trp Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
Arg 100 105 110Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser
Asp Glu Gln 115 120 125Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu
Leu Asn Asn Phe Tyr 130 135 140Pro Arg Glu Ala Lys Val Gln Trp Lys
Val Asp Asn Ala Leu Gln Ser145 150 155 160Gly Asn Ser Gln Glu Ser
Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 165 170 175Tyr Ser Leu Ser
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 180 185 190His Lys
Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 195 200
205Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210
21516666DNAArtificial Sequenceanti-CD19Fab heavy chain-nucleic
acidVH(1)..(372)CH(373)..(666) 16caggtgcagc tggtccagtc cggggcagaa
gtgaagaaac caggagcctc tgtgaaagtc 60agttgtaagg cttcaggcta taccttcatc
tcttactgga tgaactgggt gagacaggca 120ccaggacagg gactggagtg
gatgggacag atttggcctg gcgatgggga caccaactat 180aatcagctgc
tgaaggataa agcaactctg accgccgaca agagcgcctc caccgcttac
240atggagctgt ctagtctgcg aagcgaagac acagctgtgt actattgcgc
acggagagaa 300accacaactg tgggccgcta ctattacgca atggattact
ggggacaggg cacactggtg 360actgtctcaa gcgctagcac caagggccca
tcggtcttcc ccctggcacc ctcctccaag 420agcacctctg ggggcacagc
ggccctgggc tgcctggtca aggactactt ccccgaaccg 480gtgacggtgt
cgtggaactc aggcgccctg accagcggcg tgcacacctt cccggctgtc
540ctacagtcct caggactcta ctccctcagc agcgtggtga ccgtgccctc
cagcagcttg 600ggcacccaga cctacatctg caacgtgaat cacaagccca
gcaacaccaa ggtggacaag 660aaagtt 66617222PRTArtificial Sequence4.
anti-CD19Fab heavy chain-amino acidVH(1)..(127)CH(128)..(222) 17Gln
Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10
15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ile Ser Tyr
20 25 30Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp
Met 35 40 45Gly Gln Ile Trp Pro Gly Asp Gly Asp Thr Asn Tyr Asn Gln
Leu Leu 50 55 60Lys Asp Lys Ala Thr Leu Thr Ala Asp Lys Ser Ala Ser
Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95Ala Arg Arg Glu Thr Thr Thr Val Gly Arg
Tyr Tyr Tyr Ala Met Asp 100 105 110Tyr Trp Gly Gln Gly Thr Leu Val
Thr Val Ser Ser Ala Ser Thr Lys 115 120 125Gly Pro Ser Val Phe Pro
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly 130 135 140Gly Thr Ala Ala
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro145 150 155 160Val
Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr 165 170
175Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val
180 185 190Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile
Cys Asn 195 200 205Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
Lys Val 210 215 22018639DNAArtificial Sequenceanti-CD20Fab light
chain-nucleic acidVL(1)..(321)CL(322)..(639) 18cagatcgtgc
tgagccagag ccccgccatc ctgagcgcca gccccggcga gaaggtgacc 60atgacctgca
gggccagcag cagcgtgagc tacatccact ggttccagca gaagcccggc
120agcagcccca agccctggat ctacgccacc agcaacctgg ccagcggcgt
gcccgtgagg 180ttcagcggca gcggcagcgg caccagctac agcctgacca
tcagcagggt ggaggccgag 240gacgccgcca cctactactg ccagcagtgg
accagcaacc cccccacctt cggcggcggc 300accaagctgg agatcaagag
gacggtggct gcaccatctg tcttcatctt cccgccatct 360gatgagcagt
tgaaatctgg aactgcctct gttgtgtgcc tgctgaataa cttctatccc
420agagaggcca aagtacagtg gaaggtggat aacgccctcc aatcgggtaa
ctcccaggag 480agtgtcacag agcaggacag caaggacagc acctacagcc
tcagcagcac cctgacgctg 540agcaaagcag actacgagaa acacaaagtc
tacgcctgcg aagtcaccca tcagggcctg 600agctcgcccg tcacaaagag
cttcaacagg ggagagtgt 63919213PRTArtificial Sequenceanti-CD20Fab
light chain-amino acidVL(1)..(107)CL(108)..(213) 19Gln Ile Val Leu
Ser Gln Ser Pro Ala Ile Leu Ser Ala Ser Pro Gly1 5 10 15Glu Lys Val
Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Ile 20 25
30His Trp Phe Gln Gln Lys Pro Gly Ser Ser Pro Lys Pro Trp Ile Tyr
35 40 45Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Val Arg Phe Ser Gly
Ser 50 55 60Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Glu
Ala Glu65 70 75 80Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Thr Ser
Asn Pro Pro Thr 85 90 95Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg
Thr Val Ala Ala Pro 100 105 110Ser Val Phe Ile Phe Pro Pro Ser Asp
Glu Gln Leu Lys Ser Gly Thr 115 120 125Ala Ser Val Val Cys Leu Leu
Asn Asn Phe Tyr Pro Arg Glu Ala Lys 130 135 140Val Gln Trp Lys Val
Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu145 150 155 160Ser Val
Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser 165 170
175Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala
180 185 190Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys
Ser Phe 195 200 205Asn Arg Gly Glu Cys 21020654DNAArtificial
Sequenceanti-CD20Fab heavy chain-nucleic
acidVH(1)..(360)CH(361)..(654) 20caggtgcagc tgcagcagcc cggcgccgag
ctggtgaagc ccggcgccag cgtgaagatg 60agctgcaagg ccagcggcta caccttcacc
agctacaaca tgcactgggt gaagcagacc 120cccggcaggg gcctggagtg
gatcggcgcc atctaccccg gcaacggcga caccagctac 180aaccagaagt
tcaagggcaa ggccaccctg accgccgaca agagcagcag caccgcctac
240atgcagctga gcagcctgac cagcgaggac agcgccgtgt actactgcgc
caggagcacc 300tactacggcg gcgactggta cttcaacgtg tggggcgccg
gcaccaccgt gaccgtgagc 360gctagcacca agggcccatc ggtcttcccc
ctggcaccct cctccaagag cacctctggg 420ggcacagcgg ccctgggctg
cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 480tggaactcag
gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca
540ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg
cacccagacc 600tacatctgca acgtgaatca caagcccagc aacaccaagg
tggacaagaa agtt 65421218PRTArtificial Sequenceanti-CD20Fab heavy
chain-amino acidVH(1)..(122)CH(123)..(218) 21Gln Val Gln Leu Gln
Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala1 5 10 15Ser Val Lys Met
Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Asn Met His
Trp Val Lys Gln Thr Pro Gly Arg Gly Leu Glu Trp Ile 35 40 45Gly Ala
Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe 50 55 60Lys
Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr65 70 75
80Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95Ala Arg Ser Thr Tyr Tyr Gly Gly Asp Trp Tyr Phe Asn Val Trp
Gly 100 105 110Ala Gly Thr Thr Val Thr Val Ser Ala Ser Thr Lys Gly
Pro Ser Val 115 120 125Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser
Gly Gly Thr Ala Ala 130 135 140Leu Gly Cys Leu Val Lys Asp Tyr Phe
Pro Glu Pro Val Thr Val Ser145 150 155 160Trp Asn Ser Gly Ala Leu
Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175Leu Gln Ser Ser
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190Ser Ser
Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200
205Pro Ser Asn Thr Lys Val Asp Lys Lys Val 210
21522639DNAArtificial Sequenceanti-Her2Fab light chain-nucleic
acidVL(1)..(321)CL(321)..(639) 22gatattcaga tgacccagag cccgagcagc
ctgtccgcaa gcgtcggcga ccgcgtcacg 60attacctgcc gtgcaagcca ggatgtcaac
accgcagtgg cttggtatca gcaaaaaccg 120ggtaaagcgc cgaaactgct
gatttatagt gcctcctttc tgtactctgg cgtgccgagt 180cgtttctcag
gttcgcgcag cggcaccgat tttaccctga cgatcagctc tctgcagccg
240gaagacttcg ccacgtatta ctgccagcaa cattatacca cgccgccgac
ctttggtcaa 300ggcacgaaag tcgaaattaa aacggtggct gcaccatctg
tcttcatctt cccgccatct 360gatgagcagt tgaaatctgg aactgcctct
gttgtgtgcc tgctgaataa cttctatccc 420agagaggcca aagtacagtg
gaaggtggat aacgccctcc aatcgggtaa ctcccaggag 480agtgtcacag
agcaggacag caaggacagc acctacagcc tcagcagcac cctgacgctg
540agcaaagcag actacgagaa acacaaagtc tacgcctgcg aagtcaccca
tcagggcctg 600agctcgcccg tcacaaagag cttcaacagg ggagagtgt
63923213PRTArtificial Sequenceanti-Her2Fab light chain-amino
acidVL(1)..(107)CL(108)..(213) 23Asp Ile Gln Met Thr Gln Ser Pro
Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys
Arg Ala Ser Gln Asp Val Asn Thr Ala 20 25 30Val Ala Trp Tyr Gln Gln
Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ser Ala Ser Phe
Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Arg Ser Gly
Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp
Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro 85 90 95Thr
Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Thr Val Ala Ala Pro 100 105
110Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr
115 120 125Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu
Ala Lys 130 135 140Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly
Asn Ser Gln Glu145 150 155 160Ser Val Thr Glu Gln Asp Ser Lys Asp
Ser Thr Tyr Ser Leu Ser Ser 165 170 175Thr Leu Thr Leu Ser Lys Ala
Asp Tyr Glu Lys His Lys Val Tyr Ala 180 185 190Cys Glu Val Thr His
Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe 195 200 205Asn Arg Gly
Glu Cys 21024651DNAArtificial Sequenceanti-Her2Fab heavy
chain-nucleic acidVH(1)..(357)CH(358)..(651) 24gaagtgcagc
tggttgaatc tggcggtggc ctggttcaac cgggtggctc cctgcgtctg 60tcatgtgcag
catcgggttt caacattaaa gatacctata tccactgggt tcgtcaggca
120ccgggtaaag gcctggaatg ggtcgctcgc atttacccga ccaatggtta
tacgcgttac 180gcggatagcg tgaaaggccg cttcaccatc agcgcagaca
cctctaaaaa cacggcttat 240ctgcagatga actcgctgcg tgcggaagat
acggccgttt attactgtag ccgctggggt 300ggcgacggct tttacgcaat
ggattactgg ggtcaaggca cgctggtcac ggtctcagct 360agcaccaagg
gcccatcggt cttccccctg gcaccctcct ccaagagcac ctctgggggc
420acagcggccc tgggctgcct ggtcaaggac tacttccccg aaccggtgac
ggtgtcgtgg 480aactcaggcg ccctgaccag cggcgtgcac accttcccgg
ctgtcctaca gtcctcagga 540ctctactccc tcagcagcgt ggtgaccgtg
ccctccagca gcttgggcac ccagacctac 600atctgcaacg tgaatcacaa
gcccagcaac accaaggtgg acaagaaagt t 65125217PRTArtificial
Sequenceanti-Her2Fab heavy chain-amino
acidVH(1)..(119)CH(120)..(217) 25Glu Val Gln Leu Val Glu Ser Gly
Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala
Ala Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30Tyr Ile His Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Arg Ile Tyr Pro
Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe
Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr65 70 75 80Leu Gln
Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ser
Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln 100 105
110Gly Thr Leu Val Thr Val Ser Ala Ser Thr Lys Gly Pro Ser Val Phe
115 120 125Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala
Ala Leu 130 135 140Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
Thr Val Ser Trp145 150 155 160Asn Ser Gly Ala Leu Thr Ser Gly Val
His Thr Phe Pro Ala Val Leu 165 170 175Gln Ser Ser Gly Leu Tyr Ser
Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190Ser Ser Leu Gly Thr
Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205Ser Asn Thr
Lys Val Asp Lys Lys Val 210 215266PRTArtificial Sequencelinker with
two C-terminal cycterine 26Cys Gly Gly Gly Gly Cys1 5
* * * * *